Presentation onSpectrophotometric Determination of Protein ConcentrationPresented By

Tasnim Zaman Anika

Std no-1202007

Md.Touhidul Islam

Std no.-1202011

Syed Ahmed Tasnim

Std no-1202025

Date: 20-12-15

Presentation outlineObjectivesDiscussion on 3 spectrophotometric methodsApplications

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ObjectivesDetermination of protein concentration using three spectrophotometric methods*

Three Spectrophotometric MethodsBicinchoninic acid (BCA, Smith) Method.

Bradford Method.

Direct absorbance measurement*

Principle:

The purple color resulting from this method is due to:

A] The nitrogens in peptide bonds in protein, reduce Cu2+ ions to Cu+

(a temperature dependent reaction)

B] Cu+ chelated by two molecules of BCA, to produce a

copper-BCA complex [purple color] with maximum absorption

(max) of 562 nm.*1.Bicinchoninic acid (BCA, Smith) Method:

Reaction*

*Advantages:Because the peptide backbone is involved in the reaction, the BCA assay is less sensitive to the types of amino acids in the protein.Disadvantages:The presence of reducing agents in your buffer can interfere with the dyeThe reaction takes some time to proceed

2.Bradford Method:*a colorimetric assay based on the interaction between Coomassie brilliant blue and the arginine and aromatic residues in your protein-Binding of the dye Coomassie Brilliant Blue G-250 to protein in acidic solution causes a shift in wavelength of maximum absorption (max) of the dye from 465nm to 595nm.

Vapour liquid equilibrium

Reaction*Test tubes containing:Bradford reagent alone. (max) of the dye 465nm Test tubes containing: Bradford reagent with protein added.(max) is shifted to 595nm.

*Advantages:This assay is quick, and the reagent is not affected by the presence of reducing agentsDisadvantages:If protein doesnt contain a decent number of arginine and aromatic residues, then the dye will not bind to the protein as efficiently, resulting in an underestimation of protein concentration

*Aromatic residues, like tyrosine and tryptophan, absorb UV light at 280 nmIf extinction co-efficient for specific protein is known, the absorbance can be measured in a UV/Vis spectrometerCalculate the concentration of protein using Beers law (A = elc, where l is the path length of the spectrometer)3.Direct absorbance measurement

Graphical Representation*Figure : Absorbance Vs Concentration

*Advantages:quick and doesnt require any special reagentsDisadvantages:This method relies on having an accurate extinction coefficient, which depends on the number of aromatic residues.If there arent a decent number of aromatic residues, extinction coefficient will be quite low, and a fairly concentrated sample will be needed to get a reasonable absorbance

*Applications :Determining the exact quantity of proteins in a solution is very often necessary in the biochemical practiceSpecific protein and its order can be identified by measuring protein concentration

Conclusion *All the three methods discussed here are important for different protein analysisQuantitative determination of protein is important

THANK YOU ALL

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